Project Summary Ebolavirus spp. cause a severe hemorrhagic fever known as Ebola virus disease (EVD). EVD is a serious public health concern, both as an emerging infectious disease and a potential biothreat. The 2013-2016 Ebola pandemic in West Africa brought global attention to the challenges associated with controlling an Ebola. The ongoing outbreak in the Democratic Republic of Congo, which recently reached over 1000 cases with no signs of slowing, has only confirmed the need for improved Ebola medical countermeasures. To better control Ebola outbreaks, it is imperative for healthcare workers to be able to diagnosis and isolate infected patients at the point-of-care in a timely manner. As such, the World Health Organization and other healthcare agencies have called for development of Ebola rapid diagnostic tests (RDTs). The proposed joint effort, for development a point-of-care antigen detection assay for early diagnosis of EVD, stems from a history of successful collaborations between the academic laboratory of Dr. David AuCoin at the University of Nevada, Reno (UNR) and commercial partner, InBios International, Inc. The two organizations have valuable expertise in RDT development and navigating the FDA approval process. The effort is further bolstered by assistance from collaborators at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) who will perform all high containment level (BSL4) studies. Preliminary studies for this proposal have resulted in an Ebola RDT prototype that shows a substantial improvement in sensitivity for Zaire ebolavirus. Our goal is to further improve assay sensitivity and expand detection to all EVD causing Ebolavirus spp. A comprehensive approach with clearly defined strategies will be used to support successful development of a pan-Ebola RDT with high sensitivity. Key aspects and milestones include: i) high throughput monoclonal antibody (mAb) production using multiple immunization strategies, ii) mAb characterization and RDT-based mAb screening, iii) commercial optimization and development of multiple assay prototypes, iv) determination of analytical sensitivity and specificity and v) assay validation with samples from Ebola-infected non-human primates.